N-Alkyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides and Their Analogues: Synthesis and Multitarget Biological Activity

Based on the isosterism concept, we have designed and synthesized homologous N-alkyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides (from C1 to C18) as potential antimicrobial agents and enzyme inhibitors. They were obtained from 4-(trifluoromethyl)benzohydrazide by three synthetic approaches and characterized by spectral methods. The derivatives were screened for their inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) via Ellman’s method. All the hydrazinecarboxamides revealed a moderate inhibition of both AChE and BuChE, with IC50 values of 27.04–106.75 µM and 58.01–277.48 µM, respectively. Some compounds exhibited lower IC50 for AChE than the clinically used drug rivastigmine. N-Tridecyl/pentadecyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides were identified as the most potent and selective inhibitors of AChE. For inhibition of BuChE, alkyl chain lengths from C5 to C7 are optimal substituents. Based on molecular docking study, the compounds may work as non-covalent inhibitors that are placed in a close proximity to the active site triad. The compounds were evaluated against Mycobacterium tuberculosis H37Rv and nontuberculous mycobacteria (M. avium, M. kansasii). Reflecting these results, we prepared additional analogues of the most active carboxamide (n-hexyl derivative 2f). N-Hexyl-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2-amine (4) exhibited the lowest minimum inhibitory concentrations within this study (MIC ≥ 62.5 µM), however, this activity is mild. All the compounds avoided cytostatic properties on two eukaryotic cell lines (HepG2, MonoMac6).


Introduction
The development of novel drugs involves various medicinal chemistry approaches, including the widely used isostere/bioisostere strategy [1]. A bioisostere is a molecule that results from the exchange of an original atom or a group of atoms for an alternative, roughly similar atom or group of atoms. Based on physical and/or chemical similarity, isosteric compounds may share analogous pharmacological behavior. Usually, it is a way to ameliorate disadvantageous features of current drugs and drug candidates, e.g., low activity, drug resistance, toxicity, poor pharmacokinetic profile, etc. It is also possible to establish an original bioactivity [2,3].
The development of novel drugs involves various medicinal chemistry approaches, including the widely used isostere/bioisostere strategy [1]. A bioisostere is a molecule that results from the exchange of an original atom or a group of atoms for an alternative, roughly similar atom or group of atoms. Based on physical and/or chemical similarity, isosteric compounds may share analogous pharmacological behavior. Usually, it is a way to ameliorate disadvantageous features of current drugs and drug candidates, e.g., low activity, drug resistance, toxicity, poor pharmacokinetic profile, etc. It is also possible to establish an original bioactivity [2,3].
Then, we screened novel molecules for their antimicrobial and cytostatic properties. Moreover, since many scaffolds carrying aromatic trifluoromethyl group have showed inhibition of acetyl-and butyrylcholinesterase (AChE and BuChE) [13][14][15], we tested novel hydrazides also for this bioactivity.

Chemistry
Analogously to our previous works [11,12], hydrazine-1-carboxamides were obtained using three synthetic approaches. Most of the derivatives (2b-2i, 2k, 2l, 2n, 2p-2q) were prepared from commercially available isocyanates and the hydrazide 1 in acetonitrile. This reaction (method A) is simple, quick and it provides high yields, up to quantitative ones (89%-99%). For the synthesis of Ndecyl, tridecyl and pentadecyl derivatives 2i, 2m and 2o, where the required isocyanates were not commercially available, we prepared them in situ using the corresponding amines and triphosgene in the presence of triethylamine (TEA) under a nitrogen atmosphere, followed by addition of the hydrazide 1. The yields of this method B ranged from 78% to 91%. Finally, for the synthesis of Nmethyl derivative 1a, N-succinimidyl N-methylcarbamate in the presence of a non-nucleophilic tertiary base (N,N-diisopropylethylamine, DIPEA) was used as a non-toxic and crystalline methyl isocyanate substitute (method C with a good yield of 77%). An overview of the synthetic approaches used is depicted in Scheme 2.
Based on the antimycobacterial activity of the derivative 2f, we decided to prepare several its analogues. First, we investigated the 1,2-diacylhydrazines 3 (Scheme 3), i.e., derivative with no secondary amine group. The N´-hexyl derivative 3a was prepared via direct acylation from the 1 and hexanoyl chloride in the presence of base (TEA; yield 79%). In order to investigate the length of the Scheme 1. Design of the 1,2-diacylhydrazines 2 based on 4-(trifluoromethyl)hydrazide 1 scaffold.
Then, we screened novel molecules for their antimicrobial and cytostatic properties. Moreover, since many scaffolds carrying aromatic trifluoromethyl group have showed inhibition of acetyl-and butyrylcholinesterase (AChE and BuChE) [13][14][15], we tested novel hydrazides also for this bioactivity.

Chemistry
Analogously to our previous works [11,12], hydrazine-1-carboxamides were obtained using three synthetic approaches. Most of the derivatives (2b-2i, 2k, 2l, 2n, 2p-2q) were prepared from commercially available isocyanates and the hydrazide 1 in acetonitrile. This reaction (method A) is simple, quick and it provides high yields, up to quantitative ones (89-99%). For the synthesis of N-decyl, tridecyl and pentadecyl derivatives 2i, 2m and 2o, where the required isocyanates were not commercially available, we prepared them in situ using the corresponding amines and triphosgene in the presence of triethylamine (TEA) under a nitrogen atmosphere, followed by addition of the hydrazide 1. The yields of this method B ranged from 78% to 91%. Finally, for the synthesis of N-methyl derivative 1a, N-succinimidyl N-methylcarbamate in the presence of a non-nucleophilic tertiary base (N,N-diisopropylethylamine, DIPEA) was used as a non-toxic and crystalline methyl isocyanate substitute (method C with a good yield of 77%). An overview of the synthetic approaches used is depicted in Scheme 2.
All the compounds 2-4 (Table 1) were characterized by their 1 H-and 13 C-NMR and infrared spectra and melting points. Additionally, the purity was checked by thin-layer chromatography (TLC) and elemental analysis. Based on the antimycobacterial activity of the derivative 2f, we decided to prepare several its analogues. First, we investigated the 1,2-diacylhydrazines 3 (Scheme 3), i.e., derivative with no secondary amine group. The N -hexyl derivative 3a was prepared via direct acylation from the 1 and hexanoyl chloride in the presence of base (TEA; yield 79%). In order to investigate the length of the acyl chain, also N -hexadecanoyl derivative 3b was synthesized using carbodiimide (EDAC)/1-hydroxybenzotriazole (HOBt)-mediated coupling (97%).
All the compounds 2-4 (Table 1) were characterized by their 1 H-and 13 C-NMR and infrared spectra and melting points. Additionally, the purity was checked by thin-layer chromatography (TLC) and elemental analysis.

Inhibition of Acetyl-and Butyrylcholinesterase
Newly synthesized N-alkyl-2- [4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides 2a-2q were evaluated for their in vitro potency to inhibit AChE from electric eel (EeAChE) and BuChE from equine serum (EqBuChE) using modified Ellman's spectrophotometric method [16]. The results (Table 1 and Figure 1) were compared with those determined for rivastigmine, a clinically used drug for treatment of various dementia. From chemical point of view, it is an aromatic carbamate-based dual inhibitor of both cholinesterases. The efficacy of inhibitors is expressed as IC50, i.e., the concentration causing 50% inhibition of the enzyme activity. Based on IC50 values for both enzymes, selectivity indexes (SI) as the ratio of IC50 for BuChE/IC50 for AChE to quantify the preference for AChE were calculated (Table 1). The results were compared with those obtained for rivastigmine, an established carbamate used in the therapy of not only Alzheimer's and Parkinson's dementias due to its cholinergic effect. From molecular pharmacology point of view, this drug belongs to dual acylating pseudo-irreversible inhibitors of both AChE and BuChE. Scheme 4. Synthesis of 1,3,4-oxadiazole-2-amine 4 (Ph3P: triphenylphosphine; (BrCl2C)2: 1,2-dibromo-1,1,2,2-tetrachloroethane).

Inhibition of Acetyl-and Butyrylcholinesterase
Newly synthesized N-alkyl-2- [4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides 2a-2q were evaluated for their in vitro potency to inhibit AChE from electric eel (EeAChE) and BuChE from equine serum (EqBuChE) using modified Ellman's spectrophotometric method [16]. The results (Table 1 and Figure 1) were compared with those determined for rivastigmine, a clinically used drug for treatment of various dementia. From chemical point of view, it is an aromatic carbamate-based dual inhibitor of both cholinesterases. The efficacy of inhibitors is expressed as IC50, i.e., the concentration causing 50% inhibition of the enzyme activity. Based on IC50 values for both enzymes, selectivity indexes (SI) as the ratio of IC50 for BuChE/IC50 for AChE to quantify the preference for AChE were calculated (Table 1). The results were compared with those obtained for rivastigmine, an established carbamate used in the therapy of not only Alzheimer's and Parkinson's dementias due to its cholinergic effect. From molecular pharmacology point of view, this drug belongs to dual acylating pseudo-irreversible inhibitors of both AChE and BuChE. AChE and BuChE inhibition are expressed as the mean ± SD (n = three independent experiments). The three lowest IC50 values for each enzyme are given in bold.
Generally, with only one exception (the nonyl derivative 2i), the hydrazine-1-carboxamides 2 are stronger inhibitors of AChE. Focusing on this enzyme, IC50 values were found in a close range of 27.04 (2o)-106.75 (2i) µM. The values of ten compounds (2a, 2d-2f, 2j-2o) are superior to the IC50 for rivastigmine (56.10 µM), the other two (2b and 2g) produced comparable in vitro activity. By comparison with the parent hydrazide 1, the majority of its carboxamides (2a, 2b, 2d-2g, and 2j-2o) showed an improved inhibition of AChE; thus, this structural modification can be considered as  AChE and BuChE inhibition are expressed as the mean ± SD (n = three independent experiments). The three lowest IC50 values for each enzyme are given in bold.

Inhibition of Acetyl-and Butyrylcholinesterase
Newly synthesized N-alkyl-2- [4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides 2a-2q were evaluated for their in vitro potency to inhibit AChE from electric eel (EeAChE) and BuChE from equine serum (EqBuChE) using modified Ellman's spectrophotometric method [16]. The results (Table 1 and Figure 1) were compared with those determined for rivastigmine, a clinically used drug for treatment of various dementia. From chemical point of view, it is an aromatic carbamate-based dual inhibitor of both cholinesterases. The efficacy of inhibitors is expressed as IC 50 , i.e., the concentration causing 50% inhibition of the enzyme activity. Based on IC 50 values for both enzymes, selectivity indexes (SI) as the ratio of IC 50 for BuChE/IC 50 for AChE to quantify the preference for AChE were calculated ( Table 1). The results were compared with those obtained for rivastigmine, an established carbamate used in the therapy of not only Alzheimer's and Parkinson's dementias due to its cholinergic effect. From molecular pharmacology point of view, this drug belongs to dual acylating pseudo-irreversible inhibitors of both AChE and BuChE.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 17 they can bind into a narrow aromatic gorge, thus preventing access of the substrate to the catalytic triad [18]. Based on the alkyl length, a change of fitting into enzyme can occur. The alkyls that are flexible may also adopt a conformation that allows a better interaction with the enzyme.
All the derivatives 2 act as dual inhibitors of both cholinesterase enzymes. With an exception of N-nonyl carboxamide 2i, the derivatives produced more intense inhibition of AChE. We used selectivity index for its quantification. Some of short and intermediate alkyls led to a comparatively balanced inhibition of both cholinesterases (SI ≤ 1.5; propyl 2c, from pentyl 2e to octyl 2h). Contrarily, four derivatives led to a significantly preferential inhibition of AChE (SI > 5; 2l-2o). Notably, an escalated AChE selectivity is related to longer alkyls (from C10 to C15). Then, we screened an inhibition of cholinesterases caused by three analogues synthesized for their potential antimycobacterial activity primarily (3a, 3b and 4). These derivatives produced identical or decreased inhibition of AChE when compared to the parent hydrazide 1 (67.1-91.9 µM vs. 69.4 µM). On the other hand, all of them are more potent inhibitors of BuChE (IC50 values from 110 to 148.6 µM) with similar selectivity indexes. The shorter carbon chain (C6) is preferred over long one (C16). In general, the amides 3 and the oxadiazole 4 do not overcome the in vitro effect of the hydrazinecarboxamides 2.

Molecular Docking Study
In order to presume possible binding mode of the prepared molecules with human AChE (pdb code 4PQE) and BChE (pdb code 1POI), molecular modelling study was performed. The most potent compound 2o and the third most active derivative 2a against AChE, were chosen as representatives for thorough investigation of ligand-enzyme interactions in the active site of AChE. Similarly, the carboxamide 2e, being the most potent molecule against BuChE, was used for the determination of the binding mode in BuChE.
The best docking pose of 2a in AChE showed the molecule of the ligand placed deep within the cavity, in a close proximity to the active site triad (Figure 2). A significant amount of H-bonds (with Ser125, Tyr124, Trp86 and Asp74) indicates highly favorable orientation of 2a. Additionally, π-π stacking with Trp86 further stabilizes the ligand-enzyme non-covalent complex. Also, the carboxamide 2o binds closely to the catalytic triad at the bottom of the cavity (Figure 3) in a similar manner. There are three hydrogen bonds (with Ser125, Tyr124 and Asp74) and, in addition, this binding mode is stabilized by π-π stacking interaction with Trp86. The long tridecyl chain is heading Generally, with only one exception (the nonyl derivative 2i), the hydrazine-1-carboxamides 2 are stronger inhibitors of AChE. Focusing on this enzyme, IC 50 values were found in a close range of 27.04 (2o)-106.75 (2i) µM. The values of ten compounds (2a, 2d-2f, 2j-2o) are superior to the IC 50 for rivastigmine (56.10 µM), the other two (2b and 2g) produced comparable in vitro activity. By comparison with the parent hydrazide 1, the majority of its carboxamides (2a, 2b, 2d-2g, and 2j-2o) showed an improved inhibition of AChE; thus, this structural modification can be considered as successful increasing the activity by up to 2.6 times (1 vs. 2o and 2m).
Of course, in these analogues the length of N-alkyl chain is the only structural factor influencing the inhibitory properties. Interestingly, the elongation of the substituent decreases the activity (from C 1 to C 3 , 31.2 and 82.3 µM, respectively). Then a drop of IC 50 value was observed (IC 50 of C 4 = 38.6 µM) followed by analogous gradual reduction of the inhibitory properties up to the global minimum of 106.8 µM (2i). Next three compounds (decyl, undecyl and dodecyl 2j-2l) exhibited similar improved activity around 40-50 µM, followed by even more efficient tetradecyl (2n) and especially the best tridecyl 2m and pentadecyl 2o derivatives (IC 50 28.9 and 27.0 µM, respectively). Then, IC 50 values are increasing slowly again (up to C 18 with 72.31 µM). Starting from C 10 to C 16 , even-odd effect can be observed favoring odd alkyls. These structure-activity relationships and trends are depicted in Figure 1.
Analyzing results of BuChE inhibition, somewhat different results and structure-activity relationships were found. Overall, IC 50 values for BuChE were higher and in a broader concentration range from 58.0 µM (N-pentyl molecule 2e) up to 277.5 µM (the most active AChE inhibitor 2m). None of the derivatives 2 exhibited more potent inhibition than rivastigmine (38.4 µM). Notably, twelve carboxamides showed an improved inhibition of the parent 4-CF 3 -benzohydrazide 1 (2a-2k, 2p, 2q), even up to 3.5 times (1 vs. 2e). Clearly, N-n-alkyl from butyl to nonyl (2d-2i) is essential for enhanced BuChE inhibition with an optimum of 5-7 carbons (Figure 1). The gradual diminishing of the enzyme inhibition potency was observed for these extending chain length: from C 1 to C 3 , C 5 → C 8 , C 9 → C 10 , C 11 → C 13 .
Analogous "oscillating" activity without one dominant trend depending on alkyl chain length has been described previously, e.g., by Imramovský et al. [17]. There are many factors influencing inhibition of AChE and BuChE, not solely the length of n-alkyl chain, e.g., such as lipophilicity, electronic and steric effects. Moreover, inhibitors of AChE can interact with more enzyme "subsites", either with one or with more at once. They may interfere with peripheral anionic site, catalytic esteratic subsite (competitively, irreversibly or pseudo-irreversibly), anionic site in the active site, or they can bind into a narrow aromatic gorge, thus preventing access of the substrate to the catalytic triad [18]. Based on the alkyl length, a change of fitting into enzyme can occur. The alkyls that are flexible may also adopt a conformation that allows a better interaction with the enzyme.
All the derivatives 2 act as dual inhibitors of both cholinesterase enzymes. With an exception of N-nonyl carboxamide 2i, the derivatives produced more intense inhibition of AChE. We used selectivity index for its quantification. Some of short and intermediate alkyls led to a comparatively balanced inhibition of both cholinesterases (SI ≤ 1.5; propyl 2c, from pentyl 2e to octyl 2h). Contrarily, four derivatives led to a significantly preferential inhibition of AChE (SI > 5; 2l-2o). Notably, an escalated AChE selectivity is related to longer alkyls (from C 10 to C 15 ).
Then, we screened an inhibition of cholinesterases caused by three analogues synthesized for their potential antimycobacterial activity primarily (3a, 3b and 4). These derivatives produced identical or decreased inhibition of AChE when compared to the parent hydrazide 1 (67.1-91.9 µM vs. 69.4 µM). On the other hand, all of them are more potent inhibitors of BuChE (IC 50 values from 110 to 148.6 µM) with similar selectivity indexes. The shorter carbon chain (C 6 ) is preferred over long one (C 16 ). In general, the amides 3 and the oxadiazole 4 do not overcome the in vitro effect of the hydrazinecarboxamides 2.

Molecular Docking Study
In order to presume possible binding mode of the prepared molecules with human AChE (pdb code 4PQE) and BChE (pdb code 1POI), molecular modelling study was performed. The most potent compound 2o and the third most active derivative 2a against AChE, were chosen as representatives for thorough investigation of ligand-enzyme interactions in the active site of AChE. Similarly, the carboxamide 2e, being the most potent molecule against BuChE, was used for the determination of the binding mode in BuChE.
The best docking pose of 2a in AChE showed the molecule of the ligand placed deep within the cavity, in a close proximity to the active site triad (Figure 2). A significant amount of H-bonds (with Ser125, Tyr124, Trp86 and Asp74) indicates highly favorable orientation of 2a. Additionally, π-π stacking with Trp86 further stabilizes the ligand-enzyme non-covalent complex. Also, the carboxamide 2o binds closely to the catalytic triad at the bottom of the cavity (Figure 3) in a similar manner. There are three hydrogen bonds (with Ser125, Tyr124 and Asp74) and, in addition, this binding mode is stabilized by π-π stacking interaction with Trp86. The long tridecyl chain is heading out of the gorge (forming hydrophobic interactions with Leu76, Leu289 and Trp286), thereby hindering access of AChE to the active site.   Concerning BuChE, all the presented compounds displayed a similar orientation in the active site of BuChE, regardless of the growing size of their molecules. This may be due to a relatively spacious cavity of BuChE compared to the one in AChE. However, the derivative 2e displayed only a few detectable interactions, namely H-bond with Tyr332 and CF-π with Trp231 ( Figure 4). Nevertheless, the ligand possesses the ability to block the catalytic triad completely.   Concerning BuChE, all the presented compounds displayed a similar orientation in the active site of BuChE, regardless of the growing size of their molecules. This may be due to a relatively spacious cavity of BuChE compared to the one in AChE. However, the derivative 2e displayed only a few detectable interactions, namely H-bond with Tyr332 and CF-π with Trp231 ( Figure 4). Nevertheless, the ligand possesses the ability to block the catalytic triad completely. Concerning BuChE, all the presented compounds displayed a similar orientation in the active site of BuChE, regardless of the growing size of their molecules. This may be due to a relatively spacious cavity of BuChE compared to the one in AChE. However, the derivative 2e displayed only a few detectable interactions, namely H-bond with Tyr332 and CF-π with Trp231 ( Figure 4). Nevertheless, the ligand possesses the ability to block the catalytic triad completely.
For most compounds, the exact determination of minimum inhibitory concentrations (MIC) was not feasible due to their limited solubility in the testing medium (evidenced by precipitation and/or turbidity); their MIC exceeded 250 µM. None of the derivatives 1-4 was capable of M. avium inhibition. M. tuberculosis and both strains of M. kansasii were inhibited by six compounds. Among primarily designed derivatives 2, the derivatives with the shortest alkyl chains (2a and 2b) were negligibly active (MIC of 1000 µM). On the other hand, N-hexyl derivative 2f exhibited a uniform MIC of 250 µM. Thus, we synthesized and evaluated its analogues 3 and 4. The cyclization of the carboxamide 2f to 1,3,4-oxadiazole 4 retained potency for M. tuberculosis (MIC of 125-250 µM) and improved activity against M. kansasii mildly (62.5-250 µM). N´-hexanoylhydrazide 3a exhibited lower MIC for M. tuberculosis (125 µM), but this modification hampered efficacy against M. kansasii (>250 µM).
Drawing a comparison between the parent hydrazide 1 [4] and its analogues, two derivatives were superior (3a, 4) and the activity of 2f was identical. All of these compounds produced lower MIC against M. kansasii. The first-line antituberculotic hydrazide drug isoniazid (INH), involved for comparison, exhibited significantly higher growth inhibition of M. tuberculosis (1 µM) and the isolate of M. kansasii from a patient. On the other hand, the hydrazide 2f and the oxadiazole 4 were superior against M. kansasii 235/80.
Despite these structure-activity relationships described, the activity against both tuberculous and nontuberculous mycobacteria is significantly lower than in the case of original N-alkyl-2isonicotinoylhydrazine-1-carboxamides, i.e., derivatives of INH. That is why no MIC for multidrugresistant M. tuberculosis strains were determined.

Cytostatic Properties
Since compounds containing trifluoromethyl group has been known for their toxic action on eukaryotic cells and approved as anticancer drugs [19], we investigated cytostatic properties of the
For most compounds, the exact determination of minimum inhibitory concentrations (MIC) was not feasible due to their limited solubility in the testing medium (evidenced by precipitation and/or turbidity); their MIC exceeded 250 µM. None of the derivatives 1-4 was capable of M. avium inhibition. M. tuberculosis and both strains of M. kansasii were inhibited by six compounds. Among primarily designed derivatives 2, the derivatives with the shortest alkyl chains (2a and 2b) were negligibly active (MIC of 1000 µM). On the other hand, N-hexyl derivative 2f exhibited a uniform MIC of 250 µM. Thus, we synthesized and evaluated its analogues 3 and 4. The cyclization of the carboxamide 2f to 1,3,4-oxadiazole 4 retained potency for M. tuberculosis (MIC of 125-250 µM) and improved activity against M. kansasii mildly (62.5-250 µM). N´-hexanoylhydrazide 3a exhibited lower MIC for M. tuberculosis (125 µM), but this modification hampered efficacy against M. kansasii (>250 µM).
Drawing a comparison between the parent hydrazide 1 [4] and its analogues, two derivatives were superior (3a, 4) and the activity of 2f was identical. All of these compounds produced lower MIC against M. kansasii. The first-line antituberculotic hydrazide drug isoniazid (INH), involved for comparison, exhibited significantly higher growth inhibition of M. tuberculosis (1 µM) and the isolate of M. kansasii from a patient. On the other hand, the hydrazide 2f and the oxadiazole 4 were superior against M. kansasii 235/80.
Despite these structure-activity relationships described, the activity against both tuberculous and nontuberculous mycobacteria is significantly lower than in the case of original N-alkyl-2isonicotinoylhydrazine-1-carboxamides, i.e., derivatives of INH. That is why no MIC for multidrugresistant M. tuberculosis strains were determined.

Cytostatic Properties
Since compounds containing trifluoromethyl group has been known for their toxic action on eukaryotic cells and approved as anticancer drugs [19], we investigated cytostatic properties of the Molecules 2020, 25, 2268 9 of 17 derivatives 2-4 using human hepatocellular carcinoma (HepG2) and monocyte (MonoMac6) cell lines. The compounds with shorter alkyls (2a-2d) and parent 4-(trifluoromethyl)benzohydrazide (1) as well avoided any cytostatic action for both cell lines at a concentration of 100 µM. The remaining compounds have no cytostatic effect up to 50 µM. Higher concentrations were not investigated due to solubility problems, i.e., precipitation of crystals. In sum, none of the compounds showed any cytostatic properties against these eukaryotic cells.

General
All the reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) or Penta Chemicals (Prague, Czech Republic) and they were used as received. The purity of the compounds was monitored by thin-layer chromatography (TLC). TLC plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck Millipore, Darmstadt, Germany) with UV detection (254 nm). The melting points were determined on a B-540 Melting Point apparatus (Büchi, Flawil, Switzerland) using open capillaries and they are uncorrected. Infrared spectra were recorded on a FT-IR spectrometer using the ATR-Ge method (Nicolet 6700 FT-IR, Thermo Fisher Scientific, Waltham, MA, USA) in the range of 650-4000 cm −1 . The NMR spectra were measured in  at ambient and higher (80 • C) temperature using a Varian V NMR S500 instrument (500 MHz for 1 H and 126 MHz for 13 C; Varian Corp., Palo Alto, CA, USA). The chemical shifts δ are given in ppm and were referred indirectly to tetramethylsilane via signals of DMSO-d 6 (2.50 for 1 H and 39.51 for 13 C spectra) or DMF-d 7 (2.75, 2.92 and 8.03 for 1 H, 29.76, 34.89 and 163.15 for 13 C spectra). The coupling constants (J) are reported in Hz. Elemental analysis was performed on a Vario MICRO Cube Element Analyzer (Elementar Analysensysteme, Hanau, Germany). Both calculated and found values are given as percentages.
The calculated logP values (ClogP) that are the logarithms of the partition coefficients for octan-1-ol/water, were determined using the program CS ChemOffice Ultra version 18.0 (CambridgeSoft, Cambridge, MA, USA).
The identity of the known compounds was established using NMR and IR spectroscopy. Additionally, their purity was checked by melting points measurement and elemental analysis. The compounds were considered pure if they agree within ±0.4% with theoretical values.

Synthesis of N-alkyl Hydrazine-1-carboxamides 2
Method A 4-(Trifluoromethyl)benzohydrazide (1, 204.2 mg, 1.0 mmol) was dissolved in anhydrous acetonitrile (MeCN,8 mL) and then the appropriate isocyanate (1.05 mmol) was added in one portion. The reaction mixture was stirred at the room temperature for 8 h, then stored for 2 h at −20 • C. Resulting precipitate was filtered off, washed with a small volume of MeCN and dried. The products were recrystallised from ethyl acetate if necessary.

Method B
Method B is based on generation of an appropriate isocyanate in situ. Triphosgene (bis(trichloromethyl) carbonate; 118.7 mg, 0.4 mmol) was dissolved in anhydrous dichloromethane (DCM; 5 mL) under nitrogen atmosphere and the appropriate amine (1.01 mmol) dissolved in anhydrous DCM (5 mL) was added dropwise. The mixture was stirred for 30 min at room temperature, then treated with triethylamine (TEA; 293 µL, 2.1 mmol). After 30 min, 4-(trifluoromethyl)benzohydrazide (1, 204.2 mg, 1.0 mmol) was added. The reaction mixture was stirred for 10 h at room temperature, then evaporated to dryness, treated with water (10 mL) and extracted with ethyl acetate (3 × 15 mL). The combined organic phase was dried over anhydrous sodium sulphate, filtered off and evaporated to dryness to give the final product, which was crystallised from ethyl acetate.

Synthesis of 1,2-diacylhydrazines 3
Method A 4-(Trifluoromethyl)benzohydrazide (1, 204.2 mg, 1.0 mmol) was dissolved in DCM (8 mL) together with triethylamine (1.5 mmol, 209 µL). Then, an acyl chloride (1.1 mmol) was added in one portion. The reaction mixture was stirred at the room temperature for 1 h. Then, resulted precipitate was filtered off, washed with a small volume of DCM and crystallised from ethyl acetate.
The 50% inhibitory concentration (IC 50 ) values were determined from the dose-response curves. The curves were defined using Microcal TM Origin1 version 7.6 software (OriginLab, Northampton, MA, USA). Cytostasis (%) was plotted as a function of concentration, fitted to a sigmoidal curve and, based on this curve, the half maximal inhibitory concentration (IC 50 ) value was determined representing the concentration of a compound required for 50% inhibition in vitro.

Conclusions
Based on the isostere concept, we designed and prepared a series of N-alkyl-2- [4-(trifluoromethyl) benzoyl]hydrazine-1-carboxamides using three synthetic procedures. Although they were proposed as possible isoniazid analogues, their antimycobacterial activity was predominantly low. The most active N-hexyl-2- [4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamide served as a lead structure for further structural optimization providing 1,2-diacylhydrazines and 1,3,4-oxadiazole. However, these modifications resulted in improved, but still mild antimycobacterial properties, again below expectation. Together with this antibacterial action, these compounds lack any cytostatic properties for eukaryotic cell lines despite the presence of a trifluoromethyl group.
The hydrazinecarboxamides were found to be dual inhibitors of both acetylcholinesterase and butyrylcholinesterase with IC 50 values in the micromolar range. Almost all the derivatives inhibited AChE preferentially. N-Methyl, tridecyl and pentadecyl groups contributed to the most potent inhibition of AChE, while pentyl, hexyl and heptyl substituents led to an improved activity against BuChE. Molecular docking suggested the binding mode for the hydrazine-1-carboxamides, which are placed deep in the cavity in a close proximity to the active site triad.
In general, although designed as bioisosteres, new carboxamides did not behave pharmacologically in the same way as original isoniazid derivatives, in other words, they are not bioisosteres in the strict sense, but only isosteres. We identified several structure-activity relationships including the conclusion that the modification of the parent 4-(trifluoromethyl)benzohydrazide scaffold provided more active compounds with interesting biological properties for further structural optimization.